The broad, long-term objective of this project is to establish and characterize the mechanism of action of ethanol on the GABAA receptor. The work will progress in three principal directions. First, we will establish the nature of interactions between ethanol and neuroactive steroids on the alpha1beta2gamma2 GABAA receptor. In these experiments, we will determine the range of endogenous steroids that can interact with the receptor to produce ethanol-mediated potentiation, and carry put experiments to gain insight into the molecular and kinetic mechanisms of ethanol action by using neuroactive steroids of varying effects and site-directed mutagenesis of the GABAA receptor subunits. Second, we will examine the mechanism of direct potentiating effect of ethanol (i.e., in the absence of a cofactor) on GABA activated currents from alpha4beta2delta receptors. And third, we will establish the potentiating effect of ethanol on native receptors in rodent brain slices. The specific goals of the proposed research are: (1) To establish whether endogenous neuroactive steroids and ethanol positively interact on the recombinant alpha1beta2gamma2 GABAA receptor to potentiate the receptor function. (2) To study the molecular and kinetic mechanisms of alpha1beta2gamma2 GABAA receptor modulation by ethanol. (3) To determine the mechanism of direct action of ethanol on recombinant alpha4beta2delta GABAA receptors. (4) To study the effect of ethanol on whole-cell currents in brain slices. The work will be carried out on recombinant wild type and mutant alpha1beta2gamma2 and alpha4beta-delta subunit-containing) GABAA receptors transiently expressed in HEK 293 cells, and on thalamic slices expressing native GABAA receptors. Receptor activation and its modulation by steroids and ethanol will be examined from the recordings of single-channel and whole-cell currents. The proposed research has been designed to test the hypothesis that ethanol-linked inebriation is mediated by the activation of the GABAA receptor in the central nervous system, and that receptor interaction with endogenous neuroactive steroids is a critical component in such activation of the GABAA receptor system. The results from the experiments will be used to examine the mechanism and structural requirements in the ethanol-mediated modulation of the GABAA receptor function.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AA014707-01A2
Application #
6919080
Study Section
Health Services Research Review Subcommittee (AA)
Program Officer
Twombly, Dennis
Project Start
2005-05-05
Project End
2007-04-30
Budget Start
2005-05-05
Budget End
2006-04-30
Support Year
1
Fiscal Year
2005
Total Cost
$219,938
Indirect Cost
Name
Washington University
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Akk, Gustav; Covey, Douglas F; Evers, Alex S et al. (2010) Kinetic and structural determinants for GABA-A receptor potentiation by neuroactive steroids. Curr Neuropharmacol 8:18-25
Li, P; Reichert, D E; Rodriguez, A D et al. (2008) Mechanisms of potentiation of the mammalian GABAA receptor by the marine cembranoid eupalmerin acetate. Br J Pharmacol 153:598-608
Akk, Gustav; Li, Ping; Bracamontes, John et al. (2008) Mutations of the GABA-A receptor alpha1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids. Mol Pharmacol 74:614-27
Akk, Gustav; Covey, Douglas F; Evers, Alex S et al. (2007) Mechanisms of neurosteroid interactions with GABA(A) receptors. Pharmacol Ther 116:35-57
Li, Ping; Bracamontes, John; Katona, Bryson W et al. (2007) Natural and enantiomeric etiocholanolone interact with distinct sites on the rat alpha1beta2gamma2L GABAA receptor. Mol Pharmacol 71:1582-90
Eisenman, Lawrence N; Shu, Hong-Jin; Akk, Gustav et al. (2007) Anticonvulsant and anesthetic effects of a fluorescent neurosteroid analog activated by visible light. Nat Neurosci 10:523-30
Akk, Gustav; Li, Ping; Manion, Brad D et al. (2007) Ethanol modulates the interaction of the endogenous neurosteroid allopregnanolone with the alpha1beta2gamma2L GABAA receptor. Mol Pharmacol 71:461-72
Li, Ping; Covey, Douglas F; Steinbach, Joe Henry et al. (2006) Dual potentiating and inhibitory actions of a benz[e]indene neurosteroid analog on recombinant alpha1beta2gamma2 GABAA receptors. Mol Pharmacol 69:2015-26
Akk, Gustav; Shu, Hong-Jin; Wang, Cunde et al. (2005) Neurosteroid access to the GABAA receptor. J Neurosci 25:11605-13